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Method and unit for detecting an interaction such as hybridization, bioassay plate provided with a number of such detecting units, system for detecting an interaction such as hybridization, and reagent kit

a technology of intercalator and detecting unit, which is applied in the field of intercalator detection technique using salt-containing hepes buffer, can solve the problem that the binding characteristics of intercalator cannot be easily retained for a long time, and achieve the effects of improving the accuracy of hybridization detection, high general utility, and high utility valu

Inactive Publication Date: 2006-01-26
SONY CORP
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Benefits of technology

[0013] To immobilize a probe substance in a reaction region on a substrate via an avidin-biotin linkage, for example, a positively-charged solid-phase surface (avidin layer) is arranged in the reaction region. When the positively-charged solid-phase surface is arranged in the reaction region, substances, an intercalator and the like, each of which has a negative charge, may nonspecifically adsorb. The present invention prevents the nonspecific adsorption of substances, each of which has a negative charge, to a positively-charged solid-phase surface by adjusting the concentration of the salt in the salt-containing HEPES buffer.
[0045] The interaction detecting units according to the present invention are useful in a bioassay plate such as a DNA chip. The bioassay plate, such as the DNA chip, and interaction detecting system according to the present invention are useful for the detection of an interaction such as hybridization. In addition, the combination of reagents, which are used in the above-described methods, into a kit can simplify the preparation procedure of the reagents. The present invention is, therefore, believed to be useful in industry.

Problems solved by technology

In the past detection of an interaction by the use of a bioassay plate involves a problem in that substances having a negative charge, such as nucleic acids, nonspecifically adsorb to a positively-charged surface of a solid phase in each reaction region.
Further, the binding characteristics of an intercalator can be hardly retained for a long time.

Method used

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  • Method and unit for detecting an interaction such as hybridization, bioassay plate provided with a number of such detecting units, system for detecting an interaction such as hybridization, and reagent kit
  • Method and unit for detecting an interaction such as hybridization, bioassay plate provided with a number of such detecting units, system for detecting an interaction such as hybridization, and reagent kit
  • Method and unit for detecting an interaction such as hybridization, bioassay plate provided with a number of such detecting units, system for detecting an interaction such as hybridization, and reagent kit

Examples

Experimental program
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Effect test

example 1

[0107] Buffers suited for the structural stability of an intercalator were verified by thin-layer chromatography. As the intercalator, “SYBRGreen I Nucleic Acid Gel Stain” (trade name, product of Cambrex Corporation; “SYBR” is a registered trademark of Molecular Probes, Inc.; hereinafter referred to as “SYBRGreen I”) was used. “SYBRGreen I” is a high-sensitivity color-developing reagent useful for the detection of double-stranded nucleic acids. “SYBRGreen I” exhibits sensitivity as high as 25 to 100 times of ethidium bromide, it is a composition considered to be adequate as an intercalator. It is to be noted that “SYBRGreen I” is labeled with a fluorescent colorant and permits the detection of its binding to a double-bonded nucleic acid on the basis of fluorescence. The experimental procedure will be described next.

[0108] Firstly, SYBRGreen I was diluted tenfold with the buffers shown in Table 1 to provide experimental samples (Sample 1 to Sample 7). For the preparation of the HEPE...

example 2

[0115] An investigation was conducted about the relationship between the temperature and pH of a buffer.

[0116] Provided were HEPES buffers, the pHs of which were 6.8, 7.1, 7.4 and 7.7, respectively, at room temperature (25° C.), and a Tris-EDTA buffer (pH 7.5). The pHs of the individual buffers were measured at temperatures of 35° C., 45° C., 55° C. and 65° C. The results are shown in FIG. 5.

[0117] As shown in FIG. 5, the Tris-EDTA buffer significantly varied in pH as the temperature changed. The HEPES buffers, on the other hand, underwent only small variations in pH even when their temperatures arose.

[0118] An intercalator may be impaired in its binding characteristics to a double-stranded nucleic acid and the quantity of fluorescence upon its binding to the double-stranded nucleic acid even by a very small change in the conditions. The above-described experimental results indicate that the use of a HEPES buffer makes it possible to control a pH change small even when conditions...

example 3

[0119] Verification was conducted on buffers capable of making greater the binding ratio of the binding between an intercalator and a double-stranded nucleic acid to the binding between the intercalator and a single-stranded nucleic acid. The following experimental procedure was followed.

[0120] Firstly, the buffers shown in Table 2 were provided.

TABLE 2SampleBuffer used for the dilution of SYBRGreen ISample 10.1 M HEPES, MgCl2 20 mM, pH 7.7Sample 2Phosphate buffer, NaCl 200 mM, pH 7.15Sample 3Tris-EDTA buffer, NaCl 200 mM, pH 7.8Sample 4Tris-EDTA buffer, MgCl2 20 mM, pH 7.5

[0121] Next, SYBRGreen I was diluted 10,000-fold with the buffers, respectively. A single-stranded or double-stranded nucleic acid of 30 mer was added at 100 nM and, after having been left over at room temperature for 10 minutes or 30 minutes, fluorometry was performed. The fluorometric results of those left over at room temperature for 10 minutes are shown in Table 3, and the fluorometric results of those left...

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Abstract

Disclosed is a method for the detection of an interaction between substances. According to the method, a salt-containing HEPES buffer is allowed to exist in a reaction region that provides a place of interaction for the interaction. The concentration of the salt in the salt-containing HEPES buffer may be adjusted such that one or more substances each having a negative charge, such as a probe nucleic acid, target nucleic acid and / or intercalator, can be prevented from undergoing non-specific adsorption on a positively-charged surface of a solid phase. Also disclosed are an interaction detecting unit including the reaction region and a medium existing in the reaction region and including the salt-containing HEPES buffer; a bioassay plate including a DNA chip provided with a number of such interaction detecting units; a system for detecting an interaction such as hybridization; and a reagent kit including the salt-containing HEPES buffer.

Description

BACKGROUND OF THE INVENTION [0001] This invention relates to an interaction detecting technique making use of a salt-containing HEPES buffer. More specifically, the present invention is concerned with a method and unit for detecting an interaction such as hybridization, a bioassay plate provided with a number of such detecting units, a system for detecting an interaction such as hybridization, and a reagent kit, all of which make use of a salt-containing HEPES buffer. [0002] Nowadays, bioassay plates such as DNA chips (or DNA microarrays) are used in mutation analyses of genes, SNPs (single-base polymorphisms) analyses, gene expression frequency analyses, and the like, and have begun to find utility in a wide variety of fields such as drug developments, clinical diagnoses, pharmacogenomics and forensic medicine. [0003] The term “bioassay plate” as used herein means a glass, silicon or like substrate with a wide variety of numerous detecting or probe substances (hereinafter referred ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6813C12Q1/6832C12Q2527/137
Inventor NEGISHI, MAKIKO
Owner SONY CORP
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